Promoter engineering‐mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram‐scale

Abstract

Here, we report a bienzymatic cascade to produce β-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82%-95% for the desired product. Finally, a kilogramscale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce β-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from β-amino acids with 82% yield and > 99% purity.

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

“…We further revealed that this bioinspired design facilitates the one‐step synthesis of a sitagliptin intermediate, specifically the conversion of 3‐OTfBE into 3‐ATfBA in a single one‐pot reaction (Scheme 2). This β‐amino acid is commonly synthesized through a bienzymatic cascade reaction that involves an esterase and a transaminase or a recombinant Escherichia coli strain coexpressing both enzymes [22, 23, 38] . A series of chiral 3‐substituted cyclo‐hexylamine derivatives, capsaicin analogs and nylon‐6 monomer 6‐aminohexanoic acid, to cite some significant examples, have also been synthesized by combining hydrolases with ωTAs as separate catalytic entities [39–41] .…”

Synthesis of Sitagliptin Intermediate by a Multi-Enzymatic Cascade System Using Lipase and Transaminase With Benzylamine as an Amino Donor

“…The promotor engineering strategy is applied to achieve the optimum expression level of Est PS as described in our previous study (Khobragade et al, 2021). In brief, the gene (Est PS) was cloned in the different strengths of T7 promotors in the pET15b plasmid (Chizzolini et al, 2014).…”

“…For reactions using benzylamine as an amine donor, the AHR/FDH system was used to remove the benzaldehyde. However, the system was not adopted in reactions with (S)-α-MBA used as an amine donor because the removal of acetophenone did not reportedly improve product formation (Khobragade et al, 2021). The reactions consisted of 10 mM 1, 20 mM benzylamine, 10 mg/ml CRL, 0.5 mM PLP, and 9 mg CDW / ml of each biocatalyst.…”

“…Furthermore, it can be used to perform screening of both (S)-and (R)-selective TAs. Therefore, as a continuation to our previously published work Khobragade et al, 2021), the present study was conducted to investigate the effectiveness of benzylamine as an amino donor for the synthesis of sitagliptin intermediate by using 1 as the substrate (Figure 1). First, we selected a panel of 16 in-house TAs and assessed their specificity for benzylamine.…”

“…Recently, we reported two enzymatic strategies for the synthesis of sitagliptin intermediate using TA from Ilumatobacter coccineus (TAIC) and commercial Candida rugosa lipase (CRL) in one cascade, as well as using TAIC and esterase from Pseudomonas stutzeri (Est PS) in another cascade. In both cascades, (S)-α-methylbenzylamine ((S)-α-MBA) was used as an amine donor, resulting in ∼82% conversion to the desired product Khobragade et al, 2021).…”